Identification of Two Elusive Human Ribonuclease MRP-Specific Protein Components

Figures

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  Figure 1.. RNase MRP/P complexes and use of the Fireworks fluorescence system to detect their deficiency in single human cells. RNase MRP and RNase P share most of their protein components. Human RNase MRP has no known unique protein components. A-D. Known components and structures of S. cerevisiae and human RNase MRP and RNase P. A. PDB: 7C7921. B. PDB: 6AGB20. C. The structure of human RNase MRP remains unknown9. D. PDB: 6AHR23. E. Knockouts of the human RNase MRP/P-specific RNA genes, RMRP and RPPH1, induce right-and-down fluorescence shift in the Fireworks cell line F. Knockout of the NMD factor UPF1 induces a rightward fluorescence shift in the Fireworks cell line, as reported previously30. G. Schematic of the different responses of the Fireworks cells to (i) NMD inhibition and (ii) translation inhibition, which suppresses NMD due to NMD dependence on active translation31–35. (Also see Fig. S1).

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  Figure 2.. Genome-wide iterative forward genetic screening identified components of RNase MRP and RNase P, as well as two poorly characterized genes. A. Schematic of the customized lentiviral genome-wide CRISPR-Cas9 sgRNA omission library used in the screening. B. FACS-sorting rounds of iterative genome-wide screening enrich Fireworks cells in the right-and-down blue sorting gate. C. Enrichment of two poorly characterized genes c3orf17, c18orf21, and shared components of RNase MRP/P. (Also see Fig. S2 and Supplemental Data S1.)

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  Figure 3.. Human RPP24 and RPP64 are required for the cleavage of endogenous substrates of RNase MRP but not RNase P. A. Knockouts of RPP24, RPP64, and components of RNase MRP/P produce right-and-down shift in fluorescence of the Fireworks cells. B. Knockouts of RPP24, RPP64, and RNase MRP components result in pre-rRNA processing defect at ITS1 site 2. C. Unlike knockouts of RNase P-specific and RNase P/MRP-shared components, knockouts of RPP24 and RPP64 do not cause a defect in the cleavage of tRNA-like mascRNA of the endogenous lncRNA MALAT1. RT-qPCRs are described in Methods Details; data are presented as means of at least n=3 replicates; error bars represent standard deviation. D. Analysis of CRISPR knockouts (obtained as shown in Fig. S3A) used in panels A-C, using Illumina sequencing of the sgRNA-targeted genomic sites. (Also see Fig. S3 and Supplemental Data S4)

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  Figure 4.. Human Rpp24 and Rpp64 specifically associate with RNA MRP and exhibit structural similarity to yeast RNase MRP-specific components. A. FLAG-tagged human Rpp24 and Rpp64 specifically co-purify the only known RNase MRP-specific component, RNA MRP, but not the RNase P-specific RNA component, RNA H1 from HEK293T cells. Fold enrichment of RNA MRP relative to “No ORF” negative control in the FLAG-purified fractions of Rpp24, Rpp64, Rpp25, Rpp21, Mettl1, and “No ORF control” are 131.2±27.4, 246.3±33.4, 191.0±23.4, 46.08±6.20, 0.999±0.62, and 1.00±0.21, respectively. Fold enrichment of RNA H1 relative to “No ORF” negative control in the FLAG-purified fractions of Rpp24, Rpp64, Rpp25, Rpp21, Mettl1, and “No ORF control” are 0.94±0.21, 0.97±0.21, 331.1±51.3, 640.9±75.3, 0.86±0.18, and 1.00±0.19, respectively. (Shown as means±SD.) Mettl1-FLAG36 represents a non-interacting negative control. B-E. Human Rpp64 and Rpp24 exhibit predicted structural similarities to the key pre-rRNA substrate recognition region of Rmp1, and the C2C2 zinc finger region of Snm1, respectively, in the cryo-EM structure of S. cerevisiae RNase MRP. B. PDB: 7C7A21. C. AlphaFold DB: AF-Q6NW34-F1-v454. D. PDB: 7C7A21. E.…